The Epstein-Barr virus (EBV) infects human B-lymphocytes and usually establishes a latent infection in them. In vivo and in vitro, the latently infected cells are induced to proliferate. Interestingly, the viral genome is maintained in these cells as a plasmid which is both replicated conservatively during S-phase and maintained efficiently at a stable copy number (1, 2, 3, 4). Only one of the latent viral gene products, EBNA1, and a small (1.8 kbp) cis-acting element (oriP) are required to recapitulate faithful plasmid replication in human and some other cells (5, 6, 7). Replication of oriP plasmids provides a useful model for studying control of initiation of replication, segregation of replicated DNAs, and maintenance of those DNAs in the mammalian nucleus.
Dimers of EBNA1 bind specifically to degenerate, 20 bp-sequences of DNA (8). The carboxy-terminal one-third of EBNA1 contains the residues sufficient for both dimerization and DNA-binding (FIG. 1) (9, 10, 11, 12). (By “dimerization of EBNA1” we refer to protein:protein interactions between the DNA-binding domain of EBNA1, and not to protein:protein interactions between linking domains.) The EBV genome contains twenty-six identified sites to which EBNA1 binds (13). Twenty-four of these sites are within two clusters which comprise oriP (5). Twenty sites with a high affinity for EBNA1 are embedded within a series of 30 bp repeats, termed the family of repeats (FR). The dyad symmetry element (DS), which is located 1 kbp away from FR contains four binding-sites for EBNA1 with lower affinity than those in FR, two of which are part of a 65 base pair dyad (5, 14, 8, 15, 29). The DS is required for replication of oriP and is the site at which or close to which DNA synthesis initiates (Gahn and Schildkraut, 1989). EBNA1, when bound to FR, can activate transcription of two viral promoters, one of which is ten kbp away (16, 17, 18). The ability of EBNA1 to bind to DNA is essential for its activation of replication and transcription through oriP (19, 20).
In addition to binding to FR and DS, EBNA1 can also link them, forming a loop of the intervening DNA (21, 22). Activities of EBNA1 other than DNA-binding and DNA-linking have not been identified. EBNA1 purified from insect and mammalian cells lacks detectable helicase or ATPase activity (23, 24). EBNA1's apparent lack of enzymatic activities led several labs to search for proteins with which EBNA1 can interact. No candidates that obviously contribute to the function of EBNA1 have been identified.
One study demonstrated that no small deletion within EBNA1, other than those which affect DNA-binding, abrogates the ability of EBNA1 to activate transcription or replication (19). The authors interpreted this finding to indicate that EBNA1 contains redundant activating domains. The linking domains of EBNA1 are redundant and therefore are reasonable candidates for its activating domains. Findings in another study, which show a correlation between the presence of linking domains and the activity of derivatives of EBNA1, support this contention (25).